Bifidobacterium that naturally target the hypoxia region of the tumor and multi-functional oxygen-producing nanoparticles equipped with IR780, perfluorohexane (PFH), CBP (carboplatin), and oxygen. The probes are expected to achieve targeted and synergistic FUAS therapy and dual-mode imaging to mediate tumor diagnosis and treatment. The oxygen and drugs carried in it are accurately released after FUAS stimulation, which is expected to alleviate tumor hypoxia, avoid tumor drug resistance, improve the effect of chemotherapy, and realize FUAS combined with chemotherapy antitumor therapy. This strategy is expected to make up for the deficiencies of existing synergists, improve the effectiveness and safety of treatment, and provide the foundation for future tumor therapy progress.Focused ultrasound ablation surgery (FUAS) is a novel therapy with a wide range of potential applications. However, synergists are crucial to the therapy process due to the ultrasonic energy's attenuation properties. As a result of the complex hypoxic environment in the tumor area and many factors, the existing synergists have limitations such as weak targeting, single imaging mode, and easy tumor recurrence after treatment. Because of the above deficiencies, this study intends to construct bio-targeted oxygen production probes consisting of The online version contains supplementary material available at 10.1186/s12951-023-01901-7. Tumor precision therapy is a hot research topic at present , 2. FocuBifidobacterium and Escherichia coli, due to their unique physiological characteristics, not only have specific targeting to solid tumors but also can be used as vectors for tumor-targeted therapy [bifidobacterium, as a probiotic, is the best carrier for tumor therapy due to its biological safety. The bifidobacterium has a negative surface charge. Without altering its physiological properties, bifidobacterium is anticipated to use the electrostatic adsorption to direct cationic NPs to assemble in the tumor target area and achieve biological targeting [Another critical problem is the targeting of PFH NPs. The lack of targeting of NPs will disperse throughout the body, and the amount of retention in tumor target areas will be small, which will affect the efficacy and safety of treatment. Although tumor targeting can be achieved by labeling specific proteins, the wide variety of tumors and the microenvironment in vivo reduce the targeting ability of NPs . Given t therapy –33 and c therapy –37. It iargeting –40.With the increase of clinical demand, a single synergist cannot meet the increasing clinical demand, so the construction of NPs is becoming increasingly multi-functional. Diagnosis and treatment of tumors are closely related to imaging. While a single imaging mode cannot provide comprehensive image information, multimodal imaging will compensate for the deficiency of single imaging and provide comprehensive diagnosis and treatment information, which is of great significance to clinical treatment –43. IR78In addition to imaging monitoring, chemotherapy drugs used in combination therapy are also important. Carboplatin (CBP), a platinum analog, is a second-generation platinum-based drug. Currently, it is widely used by FDA to treat various types of cancer with chemotherapy and has an excellent anti-tumor effect due to its low cytotoxicity –48. The bifidobacterium and multi-functional oxygen-producing NPs equipped with IR780, PFH, CBP, and O2 (ICP-CLs@O2). In order to avoid the influence of chemical modification on the physiological characteristics of bifidobacterium and the impact of excessive particle size on the delivery efficiency, we chose a two-step delivery method in terms of delivery mode. Firstly, bifidobacterium was injected to proliferate at the tumor target area, and then multi-functional oxygen-producing NPs (ICP-CLs@O2) were injected. With the guidance of the electrostatic adsorption force, the self-assembly of the bio-targeted oxygen production probes in the tumor target area was completed. Under the constant monitoring of bio-targeted oxygen production probes dual-mode imaging, targeted synergistic FUAS therapy can be realized, and chemotherapy drugs and oxygen can be released at a specific point to improve tumor resistance caused by hypoxia. Finally, FUAS combined with chemotherapy can be realized as antitumor therapy and inhibit tumor growth and metastasis. This strategy is expected to compensate for tumor therapy's shortcomings and has a potential application prospect.To summarize, this study developed bio-targeted oxygen production probes in response to the limitations of existing tumor therapy, such as poor targeting, a single diagnosis and treatment image mode, and drug resistance caused by hypoxia. As illustrated in Scheme Bifidobacterium longum (ATCC 15707) was obtained from the Chongqing Key Laboratory of Biomedical Engineering. Man-Rogosa-Sharpe (MRS) broth was purchased from Qingdao Haibo Biotechnology Co. . 4,6-Diamidino-2-phenylindole (DAPI), DiI, Fluorescein isothiocyanate (FITC), and Cell Counting Kit-8 (CCK-8) assay were obtained from Beyotime Technology. (China). Chongqing East Chemical Industry Ltd., Co.  provided the chloroform (CHCl3). It was obtained from Avanti Polar Lipids in Alabama (USA) that DC-cholesterol hydrochloride (DC-CHOL), DSPE-PEG 2000-Amine, and DPPC were manufactured. IR780, perfluorohexane (PFH), and Carboplatin (CBP) were purchased from Aladdin Co., Ltd.Bifidobacterium longum (BL) was cultured anaerobically at 37 °C in MRS broth until the mid-log phase of growth. BL was collected and resuspended in PBS in the cryogenic centrifuge and adjusted to the appropriate reserve concentration .3, rotated on a rotary evaporator  at 50 °C to obtain a liposome film containing IR780 after removing the organic solvent. To completely dissolve the lipid film, deionized (DI) water was added. Under ice-water bath conditions, the mixed solution was transferred to an EP tube, and 200 µL of PFH and CBP were added and sonicated at 105W for 5 min . The unwrapped drug was removed by centrifugation at 8000 rpm . The NPs were suspended in DI water, and the steps were repeated to purify to obtain multi-functional cationic lipid nanoparticles loaded with IR780, CBP, and PFH (ICP-CLs). At last, multi-functional oxygen-producing NPs (ICP-CLs@O2) could be harvested by bubbling oxygen into ICP-CLs for 5 min. DC-cholesterol was replaced with cholesterol to synthesize ICP-Ls@O2. Fluorescent NPs can be obtained by adding dye DiI into the lipid solution.A simple one-step emulsion procedure synthesized multi-functional oxygen-producing nanoparticles (NPs) . The lipBifidobacterium longum was observed under a light microscope after gram staining. Using the NanoBrook 90Plus PALS (Brookhaven), the mean particle sizes and zeta potential of Bifidobacterium longum and ICP-CLs@O2 were tested. Transmission electron microscopy (TEM) was used to observe the internal structure of ICP-CLs@O2. With the UV–vis-NIR spectrophotometer , the absorbance of ICP-CLs@O2, CBP, and IR780 was measured, and standard curves were plotted for CBP and IR780. The oxygen concentration was measured using a portable dissolved oxygen meter  as the primary testing medium for the oxygen loading and release behavior in aqueous solutions. In order to pre-oxygenate ICP-CLs@O2, ICP-CLs were saturated with pure oxygen. To verify the effect of FUAS on drug release, the in vitro release of CBP from ICP-CLs@O2 was detected under different conditions. As a test of biocompatibility for ICP-CLs@O2, hemolysis was performed. Multi-functional oxygen-producing NPs stability was tested every four days by measuring particle size and zeta potential, which laid the foundation for follow-up in vivo testing.The morphology of BL (1 × 105 CFU/mL) and ICP-CLs@O2 (0.2 mg/mL) were mixed at a volume ratio of 1:3, then fully reacted at room temperature and let stand. The zeta potential and particle size of bio-targeted oxygen production probes were further detected.In order to construct the Bio-targeted oxygen production probes, BL and ICP-CLs@O2, verify whether bio-targeted oxygen production probes are successfully constructed. The ICP-Ls@O2 and ICP-CLs@O2 were labeled with DiI, and BL was labeled with FITC. Bio-targeted group (BL + ICP-CLs@O2) and non-targeted group (BL + ICP-Ls@O2) were observed via confocal laser scanning microscopy , judging the conglutination between BL and NPs. Flow cytometry  was used to compare the bind efficiencies of the bio-targeted and non-targeted groups.To further test the connection between 2.Murine breast cancer 4T1 cells and human umbilical vein endothelial cells (HUVECs) were provided by the Chinese Academy of Science Cell Bank, by the American Type Culture Collection (ATCC) recommendations at 37 ℃ under 5% CO6 cells suspended in 100 µL PBS solution to create the 4T1 tumor model. When the tumor diameter reached 0.5 cm, in vivo experiments were carried out.All animal treatments approved by the Chongqing Medical University Animal Ethics Committee were guided by the Chongqing Medical University Guidelines for the Care and Use of Laboratory Animals. Female BALB/c mice (6–8 weeks) were purchased from Chongqing Medical University's Laboratory Animal Center. 4T1 cells were injected subcutaneously into the mice's left flank with 1 × 102 group and ICP-CLs@O2 + FUAS group. After cell proliferation was stabilized, ICP-CLs@O2 irradiated by FUAS were added to the ICP-CLs@O2 + FUAS group, and an equal dose of ICP-CLs@O2 without FUAS was added to ICP-CLs@O2 group. In order to distinguish between live and dead cells, both groups of cells were stained with calcein-AM and observed using CLSM. Flow cytometry further quantified the cell apoptosis rate of the two groups and evaluated the chemotherapy effect of ICP-CLs@O2 on tumor cells.4T1 cells was inoculated into a confocal dish for 12 h and divided into the ICP-CLs@OBL  injections through the tail vein for three consecutive days. In the control group, mice were injected with 200 µL PBS. At post-three and post-seven days after injection, the mice in the corresponding group were euthanized, and the tumor, heart, liver, spleen, kidney, and lung tissues were extracted and homogenized. The tissue homogenates, after serially diluted, were cultured anaerobically on solid agar for 48 h. The distribution of BL in vivo tissues was assessed by observing the growth condition of colonies on the agar.Fifteen tumor-bearing mice were randomly divided into the Control group, post-3-day group, and post 7-day group. The mice in the last two groups were given BL  intravenously for three consecutive days. The mice in the non-targeted group were injected with 200 µL PBS similarly. At 7 days after injection, both groups were inject 200 µL DiI-ICP-CLs@O2 (1 mg/mL) intravenously. Mice were euthanized in two groups 24 h, 48 h, and 72 h after the DiI-ICP-CLs@O2 injection. Tumor tissue was extracted and prepared into frozen ultrathin slices. The tumor nuclei of frozen ultrathin slices were labeled with DAPI and scanned via CLSM to evaluate the targeting ability of bio-targeted oxygen production probes.30 tumor-bearing mice were randomly divided into a bio-targeted group and a non-targeted group. In the bio-targeted group, mice were injected 2 group, and BL + ICP-CLs @O2 group (bio-targeted oxygen production probes group). Tumor-bearing mice in the bio-targeted oxygen production probes group were i.v. Injected with 200 µL BL (1 × 106 CFU/mL) three times. Mice in the PBS group, ICP-CLs group, and ICP-CLs@O2 group were i.v. Injected with the same dose of PBS. Seven days after injection, the corresponding group was injected with PBS, ICP-CLs, and ICP-CLs@O2, respectively. 48 h after the second injection, FUAS treatment was administered. Then mice were given pimonidazole hydrochloride (60 mg/kg) intraperitoneally. Tumors were harvested and prepared into frozen ultrathin slices thirty minutes later. In the process of immunofluorescence staining, the mouse monoclonal antibody was used for incubation first, followed by a Dylight 649-conjugated goat anti-mice secondary antibody. In order to locate the nucleus clearly, the nuclei of tumor cells were stained with DAPI.HypoxyprobeTM-1 Kit was used to evaluate the hypoxia situations in the PBS group, ICP-CLs group, ICP-CLs@OHIF-1α expression was related to the degree of hypoxia. The experimental grouping and treatment were the same as above. The mice were sacrificed after the treatment, and the tumor tissues were extracted for the frozen section. HIF-1α rabbit antibody was used as the primary immunostain, followed by FITC-conjugated AffiniPure goat anti-rabbit secondary antibody, following DAPI staining of the tumor cell nuclei.All fluorescence images were observed using CLSM.2 and different concentrations of ICP-CLs@O2 were tested. The LB983 imaging system was used to capture the FL imaging .To test the fluorescence (FL) imaging capability of multi-functional oxygen-producing nanoparticles, 200 μL of samples with CP-CLs@O2 to identify the photoacoustic (PA) imaging capability. The Vevo LAZR PA Imaging System was used to obtain the PA imaging .The agar gel model was supplemented with various concentrations of ICP-CLs@OBL  intravenously for three consecutive days. Mice in the non-targeted group were treated with the same dose of PBS. Seven days after injection, mice in both groups were injected with 200 µL ICP-CLs@O2 through the tail vein. PA and FL Images of mice in two groups were captured at various time points after injection. To further observe the biological distribution of the bio-targeted oxygen production probes in mice, tumors and major organs of mice were extracted for FL imaging.For in vivo dual-modality imaging, mice were randomly divided into bio-targeted group and non-targeted groups with five mice each. First, mice in the bio-targeted group were injected with 2 were injected into bovine livers separately. Guided by ultrasonic images, technicians located the target area. The bovine livers target area was then treated for 3 s at 150 W with a Model-JC200 Focused Ultrasound Tumor Therapeutic System . The ultrasonic images of the target area before and after treatment were sketched, and the gray change values were calculated using Gray Val software associated with the FUAS equipment. Bovine liver after ablation with focused ultrasound was sliced to determine the maximal cross-section of the ablation area and calculate the volume of coagulated necrosis.Fresh ex vivo bovine livers were used to test the in vitro synergistic capability of multi-functional oxygen-producing nanoparticles.100 μL PBS, ICP-CLs, and ICP-CLs@O2 group, and BL + ICP-CLs@O2 group with five mice each to evaluate in vivo synergistic capability of FUAS bio-targeted oxygen production probes. For the first injection, mice in the BL + ICP-CLs@O2 group were i.v. injected with BL  for three consecutive days. Mice in other groups were i.v. injected with PBS. For the second injection, 200 μL PBS, ICP-CLs, and ICP-CLs@O2 were i.v. injected into the corresponding groups. FUAS was administered under ultrasound guidance 48 h after injection. The ultrasonic images of the tumor target area before and after treatment were sketched, and the gray change values were calculated. Twenty-four hours after FUAS, mice were euthanized, and tumors were harvested to evaluate the coagulative necrosis degree of the tumor more intuitively. The tumor tissue was incised along the acoustic beam axis to find the maximum ablation section, and TTC staining was performed . The formula calculated coagulation necrosis volume and energy efficiency factor (EEF):PBS group, ICP-CLs group, ICP-CLs@OThe mice experimental grouping and treatment process was the same as in vivo synergistic experience. After the first injection, tumor volume was measured every four days. To evaluate the relative tumor volume (RTV) before and after treatment, the initial tumor volume before the first injection was used as standardized. In order to analyze necrosis and apoptosis, mice were euthanized 17 days after injection. The tumor samples were collected and weighed before staining with H&E, PCNA, and TUNEL to evaluate the effect of antitumor therapy in vivo.2 against normal HUVECs was evaluated using the Cell Counting Kit-8 (CCK-8) assay. Blood samples were collected short, and long-term after being treated with bio-targeted oxygen production probes were injected and then detected for biochemical tests  and complete blood count  to check the biosecurity. H&E staining was performed on major organs of mice after combination therapy to evaluate the safety of the treatment.The cytotoxicity of ICP-CLs@OP (*) < 0.05 indicated statistical significance.Data were expressed as mean and standard deviation (SD) and statistically analyzed using GraphPad Prism 9.2.0. Student's t-test was used to identify the statistical significance between the two groups. One-way analysis of variance (ANOVA) was applied for multiple group comparisons. 2. ICP-CLs@O2 were clearly spherical, with black and dense cores, according to the TEM results . Due to the fact that BL used electrostatic adsorption to direct much ICP-CLs@O2 to remain in the tumor and extended the ICP-CLs@O2 stay at the tumor location. Group bio-targeted combines the tumor targeting capabilities of IR780 and biological targeting vector BL. The FL signal peaked 48 h after injection, indicating that the enrichment of probes at the tumor site was greatest at this time, which is vital for subsequent in vivo treatment. To further observe the distribution of probes in the organs, in vitro fluorescence imaging was performed on the organs , indicating that both ICP-CLs and ICP-CLs@O2 could significantly enhance FUAS ablation , and the higher the ablation efficiency, the smaller the value. Figure 2 had significantly lower EEFs than the PBS group (**P < 0.01). The results of in vitro bovine liver ablation experiments all proved that ICP-CLs@O2 could be used as a synergist in FUAS treatment.The results of bovine liver ablation showed that the liver was grayish white after ablation, the normal tissue without ablation was purplish red Fig. A. Both tion Fig. B, C. BotBL + ICP-CLs@O2 was the most significant . Moreover, the area of proliferative cells in each group ranged from large to small . No matter whether PCNA or TUNEL staining, bio-targeted oxygen production probes showed a good therapeutic effect.Figure ect Fig. C. Comparend Fig. D, E. H&E2 at various concentrations showed that ICP-CLs@O2 were safe. During the whole treatment process, mice in group BL + ICP-CLs@O2 showed a slight weight loss after BL injection and then recovered to normal soon after, with their weight fluctuating within the normal range, indicating that the bio-targeted oxygen production probes-based treatment strategy would not affect the normal growth of mice . Blood biochemical and routine blood indexes showed that the indexes of the treatment group fluctuated within the normal range, and there was no significant difference between the treatment group and the control group  The introduction of IR780 endows the bio-targeted oxygen production probes with FL and PA imaging capabilities. With the addition of two imaging modes, the original ultrasonic monitoring can be enriched, and drug delivery can be visualized. Dual-mode imaging is essential for tumor diagnosis and treatment. (3) PFH can not only carry oxygen but also enhance FUAS treatment. Meanwhile, FUAS has good tissue penetration, which can accurately control PFH to release more oxygen. By alleviating the hypoxia in the tumor area, improving the efficacy of chemotherapy, and preventing tumor recurrence and metastasis. (4) CBP, a commonly used chemotherapy drug in clinical therapy, can destroy residual tumors and achieve antitumor treatment of FUAS. It is wrapped in liposomes, enriched in the tumor target area guided by bacteria, and released after being stimulated by FUAS, reducing the damage to normal tissues, which can not only improve the effectiveness of treatment but also ensure the safety of treatment.Bio-targeted oxygen production probes have been developed to address the limitations of existing tumor therapies, such as poor targeting, single diagnostic and therapeutic image patterns, and drug resistance caused by hypoxia. The probe has the following benefits: (1) The existence of the hypoxic zone limits the effect of tumor treatment. However, unexpected effects can be achieved by using this feature and selecting the appropriate vector. The selection of probiotic The cooperation of bio-targeted oxygen production probes and FUAS therapy is anticipated to address the drawbacks of current therapies, improve efficacy while ensuring the safety of treatment, and provide a unique approach to clinical therapy.Additional file 1: Fig. S1. Light microscope image of ICP-CLs@O2 (400 magnification). Fig. S2. The standard curve of IR780. Fig. S3. The standard curve of CBP. Fig. S4. In vitro release curves of CBP. Fig. S5. Particle size of ICP-CLs@O2 over a period of 13 days. Fig. S6. Zeta potential of ICP-CLs@O2 over a period of 13 days Fig. S7. The hemolysis test of ICP-CLs@O2 at different concentrations. Fig. S8. Flow cytometry analysis binding between Bifidobacterium longum and DiI-labeled ICP-CLs@O2. Fig. S9. Cell viability assay of different concentration ICP-CLs@O2 incubated with HUVECs. Fig. S10. Variation trend of body weight in different groups of mice. Fig. S11. Hematological assay of BALB/c mice. Fig. S12. H&E staining of major organs from the control group and the treated groups.


Amblyomma sculptum is the major vector of Rickettsia rickettsii, the causative agent of the highly lethal Brazilian spotted fever. It has been shown that R. rickettsii inhibits apoptosis in both human endothelial cells and tick cells. Apoptosis is regulated by different factors, among which inhibitors of apoptosis proteins (IAPs) play a central role. In the study reported here, we selected an IAP of A. sculptum that has not yet been characterized to assess its role in cell death and to determine the effects of its gene silencing on tick fitness and R. rickettsii infection.The tick A. sculptum cell line (IBU/ASE-16) was treated with specific double-stranded RNA (dsRNA) for either IAP (dsIAP) or green fluorescent protein . The activity of caspase-3 and the exposure of phosphatidylserine were determined in both groups. In addition, unfed adult ticks, infected or not infected with R. rickettsii, were treated with either dsIAP or dsGFP and allowed to feed on noninfected rabbits. In parallel, noninfected ticks were allowed to feed on an R. rickettsii-infected rabbit. Ticks (infected or not with R. rickettsii) that remained unfed were used as a control.An R. rickettsii. Conversely, lower mortality rates were recorded in unfed ticks.Caspase-3 activity and the externalization of phosphatidylserine were significantly higher in IBU/ASE-16 cells treated with dsIAP than in those treated with dsGFP. The mortality rates of ticks in the dsIAP group were much higher than those in the dsGFP group when they were allowed to feed on rabbits, independent of the presence of A. sculptum cells. Moreover, IAP-silenced ticks experienced higher mortality rates following the acquisition of a blood meal, suggesting that feeding may trigger the activation of apoptosis in the absence of this physiological regulator. These findings indicate that IAP is a potential antigen for an anti-tick vaccine.Our results show that IAP negatively regulates apoptosis in The online version contains supplementary material available at 10.1186/s13071-023-05701-8. Amblyomma sculptum, a member of the Amblyomma cajennense species complex, is widely distributed throughout the Cerrado biome in South America, as well as in degraded areas of the Atlantic Forest in Brazil -1-propanesulfonate hydrate (CHAPS), 10% sucrose, 0.1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride (PMSF) and 2 μM pepstatin, pH 7.2]. The protein content in the resulting extracts was determined, and 50 μg of each sample was used to determine caspase-3 activity, assessed by the release of the aminomethyl coumarin (AMC) from the synthetic fluorogenic substrate Ac-DEVD-AMC, as previously described  in the midgut of P ˂ 0.05.Data were statistically analyzed using the Mann‒Whitney test with GraphPad Prism software . The differences between the groups were considered to be significant when A. sculptum IAP showed that it contains three type II BIR domains in its N-terminal region and a RING domain in its C-terminal region. These domains are conserved in the IAPs of other tick species, including the XIAP of Ixodes scapularis [The CDD analysis of the amino acid sequence of apularis , as reveP = 0.041; Fig. P = 0.0022; Fig. P = 0.0022; Fig. To determine the effects of IAP silencing on the activity of caspase-3, we incubated IBU/ASE-16 cells with either dsIAP or dsGFP Fig. . IAP expP = 0.0079; Fig. P = 0.8413; Fig. To evaluate phosphatidylserine exposure and cell viability, IBU/ASE-16 cells were treated with either dsIAP or dsGFP, labeled with annexin-V and with Live/Dead and analyzed by flow cytometry. The dsIAP group showed a significantly higher percentage of annexin V-labeled cells than the dsGFP-treated group  group when ticks were allowed to feed on rabbits, independent of the presence of ks Table . In set In contrast to the high mortality of ticks in the dsIAP groups of sets 1, 2 and 3, which were allowed to feed on rabbits, much lower mortality rates were recorded in unfed ticks in sets 4 and 5 , Acaj-59320 (P = 0.0025) and Acaj-73477 (P = 0.0025), respectively, in fed ticks.The gene expression of CDSs Acaj-73060 (IAP), Acaj-71920 (apoptotic inhibitor 5) and Acaj-59320 and Acaj-73477 (apoptotic regulators of the BCL-2 family) in ticks fed for 72 h in relation to unfed ticks was assessed. The results showed that the transcript levels of IAP Acaj-73060 were significantly lower in fed ticks than in unfed ticks  and A. sculptum (IBU/ASE-16) [R. rickettsii growth is faster in BME26 cells treated with a caspase-3 inhibitor than in control (untreated) cells while, in contrast, rickettsial growth is much slower in cells treated with staurosporine, a classic apoptosis activator. These results indicate that the inhibition of apoptosis is important for R. rickettsii survival and growth in tick cells [A. sculptum, suggesting that R. rickettsii may also control apoptosis in vivo.al cells –16 in a /ASE-16) . It shouck cells . Interesck cells  revealedOur in silico analysis of the IAP Acaj-73060 amino acid sequence showed that it possesses three type II BIR domains in the N-terminal region . A number of published studies have reported that R. rickettsii-infected ticks present higher mortality rates than uninfected ticks, suggesting a possible pathogenic effect of this bacterium on its vectors in the USA and Brazil [The mortality rates of IAP-silenced ticks allowed to feed on rabbits were much higher than those of nonsilenced ticks (treated with dsGFP), independent of the presence of es Table . On the es Table . Remarkaes Table , set 4B,es Table , set 5B,d Brazil –32. TherR. rickettsii are induced in the midgut of fed ticks, suggesting that feeding promotes the formation of a pro-oxidant environment [The detrimental effect of dsIAP administration followed by feeding suggests that the acquisition of the blood meal might activate apoptosis, causing tick death. Therefore, the gene expression of negative regulators of apoptosis in fed ticks in comparison to unfed ticks was evaluated. Our analyses showed that the expression of all CDSs, including the CDS of IAP Acaj-73060, was downregulated in fed ticks Fig. . In addiironment , 34.I. scapularis, which is similar to IAP Acaj-73060 from A. sculptum , inducing the phosphorylation of Relish, the transcription factor of this immune pathway, which is cleaved and translocated to the nucleus [A. sculptum also contains a RING domain, its E3-ubiquitin ligase activity was not evaluated in the present study. Intriguingly, A. phagocytophilum has been previously described to inhibit apoptosis in the midgut and in the salivary glands of I. scapularis [ c release [I. scapularis fitness, causing a decrease in female engorgement [Due to the high mortality rates of blood-fed ticks treated with dsIAP, it was not possible to analyze the role of IAP in rickettsial proliferation in ticks from sets 2 and 3. However, the knockdown of XIAP from tophilum . To date nucleus . This E3apularis . In the  release . XIAP siorgement . HoweverA. sculptum IAP negatively regulates apoptosis in IBU/ASE-16 cells. In addition, the gene silencing of A. sculptum IAP Acaj-73060, followed by the acquisition of a blood meal, caused a high mortality of ticks, suggesting that apoptosis regulation is essential to successful tick feeding. These results identify this protein as a target for the development of an anti-tick vaccine. Additional studies are needed to determine how the acquisition of the blood meal leads to the activation of apoptosis in the A. sculptum midgut.Our findings demonstrate that Additional file 1: Table S1. Accession numbers of IAP sequences used to perform the multiple sequence alignment and generate a phylogenetic tree are displayed in Figure 2.Additional file 2: Table S2. Specific oligonucleotides used for dsRNA synthesis and RT‒qPCR analyses.Additional file 3: Figure S1. A Multiple sequence alignment of amino acid sequences was performed using the MUSCLE method. BIR domains are shown in gray boxes, and RING domain is shown in bold letters. B Phylogenetic tree of tick IAPs. The tree was constructed with IAP amino acid sequences from 15 tick species  using the maximum likelihood method. The bar scale at the bottom indicates 10% amino acid divergence.Additional file 4: Figure S2. A Dead ticks from the dsIAP group of set 2 (uninfected ticks fed for 8 days on an R. rickettsii-infected rabbit). B Live ticks from the dsIAP group of set 4B (uninfected and unfed ticks 30 days postinjection). C Live ticks from the dsGFP group of set 4B (uninfected and unfed ticks 30 days postinjection).


Glioblastoma multiforme (GBM) is the most aggressive, malignant, and therapy-resistant tumor of the brain. Blockade therapy targeting the programmed cell death protein 1 (PD-1)/programmed death ligand (PD-L1) axis is currently under investigation for the clinical management of the GBM. This study has quantified the plasma levels of PD-L1 as a biomarker for the clinical management of GBM.n = 128) of Pakistani adult glioblastoma patients together with age- and sex-matched healthy controls was used for quantification of pre-surgery levels of plasma PD-L1. PD-L1 protein and mRNA were measured by PD-L1 platinum enzyme-linked immunosorbent assay and quantitative real-time PCR, respectively. Receiver operating characteristic (ROC) curve analysis was used to compute area under the curve (AUC) for specificity and sensitivity analyses. The Kaplan–Meier survival analysis was employed to compute overall survival.A cohort (p < 0.0001). Mean PD-L1 concentration for the GBM was found to be 48.98 ± 2.290 pg/ml compared to 27.63 ± 1.281 pg/ml for controls. Gene expression analysis showed statistically significant upregulation (p < 0.0001) of PD-L1 in blood of GBM compared to healthy controls. Plasma PD-L1 showed an AUC of 0.840  where a cutoff value higher than 46 pg/ml demonstrated 100% specificity and 57.81% sensitivity. Higher pre-surgery levels of PD-L1 were found to be associated with overall poor survival . Age, gender, and ethnic background were not found to be associated with plasma PD-L1 levels.PD-L1 protein and mRNA were significantly higher in GBM compared to the healthy controls (The study concludes that blood-based measurements of PD-L1 in GBM can be a promising prognostic marker and therapeutic target besides a rapid and relatively non-invasive screening tool for routine clinical management. Future work extending the analysis to larger cohorts through multi-center collaborations involving pre-treatment and post-treatment groups is required to fully explore the usefulness of circulating PD-L1 for effective clinical applications. GBM is also the most abundant of malignant brain tumors accounting for nearly 50% of reported gliomas in USA alone . Though Immune checkpoint inhibitors have been reported to confer anti-tumor resistance by augmenting the ability of immune system to eliminate the cancerous cells . Lately,PD-L1 has been shown to be upregulated in GBM and is particularly associated with poor prognosis, malignancy, and aggressiveness . ExtraceA soluble form of plasma PD-L1 thought to originate from PD-L1-expressing cells in the tumor microenvironment has attracted notable attention as a surrogate marker . Many re22.1Adult patients with clinically confirmed diagnosis of glioblastoma (GBM) were recruited to participate in the study from the Neurosurgery ward of the Pakistan Institute of Medical Sciences (PIMS), Islamabad after granting ethical approval from both the ethical review board (ERB) of COMSATS University Islamabad (CUI) and PIMS hospital. A written informed consent was obtained from all the study participants wherein each was informed about the research prior to collecting the blood. Healthy controls were selected with no prior history of malignant disease, diabetes, cardiovascular disease, and/or any other major illness. Controls were matched for age and gender with the malignant cohort. All the study subjects in the GBM cohort were followed up for 6 months after initial surgical excision of the tumor.To determine the efficacy and usefulness of plasma PD-L1 as a biomarker in the therapeutic management of GBM, blood samples were collected from 64 confirmed GBM patients prior to their initial surgery. Venous blood (3 ml) was drawn by the trained hospital phlebotomist in a pre-labeled ethylene diamine tetra-acetic acid (EDTA) tube to prevent clotting following standard clinical practices. A questionnaire about the disease-related information was also completed by each participant of the study for demographic analysis. The tubes were then safely transferred to the laboratory at the Department of Bioscience, CUI, in an ice box and plasma was separated. First, the blood samples were centrifuged at low speed to separate the aqueous phase and then at high speed to remove residual cells, and finally, the separated plasma was stored at −20°C until further use.2.2RNA from the pelleted cells obtained after centrifugation, described in the previous step, was extracted using TRIzol reagent (Thermo Scientific) following their protocol. RNA was dissolved in RNase-free water and was quantified using NanoDrop spectrophotometer and thereafter subjected to DNase (Thermo Scientific) treatment to remove contaminating genomic DNA. First, the DNase enzyme was added to the RNA solution and incubated for an hour at 37°C as described in the manufacturer’s assay. After incubation, samples were re-extracted using phenol-chloroform-isoamyl alcohol , and finally, the RNase-free water was added to dissolve the RNA and stored at −20°C.2.3® green master mix (Thermo Scientific) with β-actin gene as internal control and gene-specific PD-L1 primers.RNA was reverse transcribed using the first-strand cDNA synthesis kit (Thermo Scientific) as per their protocol. Reaction mix consisted of RNase inhibitor, reaction buffer, and dNTPs together with reverse transcriptase (RT) enzyme. After the completion of the reaction, cDNA was stored at −20°C until further analysis. qRT-PCR (Applied Biosystems) was performed using maxima SYBR2.4PD-L1 in the plasma was measured using a commercially available assay  according to the protocol. Briefly, wash buffer and assay buffers were prepared and human PD-L1 standard was reconstituted and dilutions were prepared. Samples and controls were added to the micro-well plate and kit-supplied biotin conjugate was then added to each of the well. This was followed by the addition of Streptavidin-horseradish peroxidase and tetramethylbenzidine (TMB) substrate solution. The reaction was terminated by the addition of the stop solution and optical density (OD) values were measured using the automated microplate reader (AMP) Platos R II at a primary wavelength of 450 nm with 620 nm as the reference wavelength. All the measurements were made in triplicate.2.5t-test or Mann–Whitney U-test based on the normality of the data. The chi-square test was used to compare categorical variables between different study groups. One-way analysis of variance (ANOVA) was used to compare more than two groups. Pearson’s or Spearman’s correlation was used to assess associations among different parameters as appropriate. Kaplan–Meier’s survival analysis was carried out to compute overall survival subsequent to initial surgery. The specificity and sensitivity of PD-L1 measurement as biomarker were determined by the area under the curve (AUC) in receiver operator characteristic (ROC) curves. Microsoft Excel (Microsoft Corporation) and GraphPad Prism V9.0  were used for all the statistical analyses.The differences between two groups were analyzed by Student’s 2.6The funding bodies did not play any role in the study design, data collection, data analysis and interpretation of the data, and writing of this manuscript. The corresponding authors were responsible for undertaking the study and ensuring the accuracy and integrity of the work. All authors have read and approved the final version.33.1The study cohort consisted of 128 individuals, of whom 64 were confirmed GBM patients and 64 were age- and sex-matched healthy controls. 3.2p < 0.0001) as shown in R2 = 0.9971) where the concentrations of known standards ranged from 9.4 pg/ml to 300 pg/ml. Pearson correlation found no statistically significant association between plasma PD-L1 levels and age  as can be seen in p = 0.2330). One-way ANOVA found no significant differences in plasma PD-L1 among different ethnic groups .PD-L1 concentration was found to be statistically significantly different between controls and GBM (3.3p < 0.0001) as shown in p = 0.6463; r = 0.058; 95% CI = −0.1901 to 0.3000). The gene expression was not found to be significantly different (p = 0.2121). Ethnicity of the study participants was also not found to be associated with PD-L1 gene expression  as can be seen in PD-L1 was significantly upregulated in peripheral blood of GBM compared to the healthy controls . AUC of ROC calculated from the measured values of circulating PD-L1 . Based on Youden index, a cutoff value of less than 46 pg/ml showed a specificity of 98.44% and a sensitivity of 59.38%. A value higher than 46 pg/ml demonstrated 100% specificity and 57.81% sensitivity. ROC curve analysis was undertaken to determine the specificity and sensitivity of the plasma levels of PD-L1 gene and protein. AUC of ROC of PD-L1 gene expression in blood  = 0.08; 95% CI = 0.04 to 0.15] between these two groups was observed  with/without contrast, and definitive pathology is confirmed by tissue biopsies. Imaging, though non-invasive, is limited by failing to distinguish actual tumor lesions from treatment-induced lesions and can often lead to diagnostic errors. Tumor biopsy, on the other hand, is highly invasive and can only offer a static glimpse into the tumor microenvironment, which, for GBM, is highly heterogeneous and ever changing with additional risk of hemorrhage and neural dysfunction , 28. LiqPD-L1 expression as a putative biomarker in GBM has been investigated with contradictory roles in prognosis mainly within tumors. Plasma PD-L1 has been shown by few other studies as a marker for primary and recurrent brain malignancies with poor prognosis and overall poor survival , 31–33. Many studies have reported the elevated levels of plasma PD-L1 in several types of malignancies with a potential to be used as a prognostic biomarker but still a baseline has not been established. It therefore is of paramount importance to characterize the expression of plasma PD-L1 in larger cohorts of different ethnicities, races, tumor types, and stages of malignancy for establishing robust and reproducible cutoff values to characterize the progress of the disease and outcome of the therapy. Also, given the role of PD-L1/PD-1 in blockade therapies, the expression has to be characterized to compensate for heterogeneity in the tumor microenvironment and measurement techniques/technologies for the effective applications of immune checkpoint inhibitors. The present study compared the differences in the expression levels of plasma PD-L1 between GBM and healthy controls recruited from local population. Blood was collected from adults with a confirmed diagnosis of GBM prior to their initial surgery, and overall survival was computed following 6 months post-surgery. To our knowledge, this is the first study describing the plasma levels of PD-L1 in a GBM cohort of Pakistani origin. The present study is limited by two important considerations: first, the study has only investigated the pre-surgery levels of plasma PD-L1 and has not contrasted it with post-surgery levels. Second, the number of patients for survival analysis was small because the study recruited participants from the largest tertiary-care hospital in the capital city where treatment to multiple brain malignancies is offered. Hence, the patient pool is highly heterogeneous with clinical presentation of different types of gliomas. This study only focused on GBM, and other gliomas were not the focus of investigation; hence, it reduced the number of eligible participants. For a comprehensive understanding of the role of plasma PD-L1 in overall survival and prognosis of GBM, future studies should compare the pre-surgery and post-surgery groups with a large sample size so that patient groups can be stratified and an optimized therapy regimen be devised.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Ethical Committee of PIMS Islamabas, Ethics Review Board of Department of Biosciences, COMSATS University Islamabad. The patients/participants provided their written informed consent to participate in this study.Conceptualization: SB and KK. Methodology: AM and AA. Software, AM and SB. Validation: SB, SNB, and KK. Formal analysis: SB, MV, and KJ. Investigation: AM and SB. Resources: SB, MV, KJ, and KK. Data curation: AM, AA, and SB. Writing—original draft preparation: AM. Writing—review and editing: SB, SNB, MV, KJ, and KK. Visualization: SB and AA. Supervision: SB and KK. Project administration: SB and KK. Funding acquisition: MV, KJ, and KK. All authors have read and agreed to the published version of the manuscript.


Few studies address the care of critically ill non-traumatic patients in the emergency department (ED). The aim of this study was to assess the epidemiology, management, and outcome of these patients.In this retrospective study, we identified and analyzed data from all consecutive adult critically ill non-traumatic ED patients treated from March 2018 to February 2019. Patient characteristics, major complaint leading to admission, out-of-hospital, and in-hospital interventions and 30-day mortality were extracted from medical records of the electronic patient data management system.During the study period, we analyzed 40,764 patients admitted to the ED. Of these, 621 (1.5%) critically ill non-traumatic patients were admitted for life-threatening emergencies to the resuscitation room . Leading problem on admission was disability/unconsciousness (D), shock (C), respiratory failure (B), airway obstruction (A), and environment problems (E) in 41%, 31%, 25%, 2%, and 1%, respectively. Out-of-hospital and in-hospital measures included: intravenous access (61% vs. 99%), 12-lead ECG (55% vs. 87%), invasive airway management (21% vs. 34%) invasive ventilation (21% vs. 34%), catecholamines (9% vs. 30%), arterial access (0% vs. 52%), and cardiopulmonary resuscitation (11% vs. 6%). The underlying diagnoses were mainly neurological (29%), followed by cardiological (28%), and pulmonological (20%) emergencies. The mean length of stay (LOS) in the resuscitation room and ED was 123 ± 122 and 415 ± 479 min, respectively. The 30-day mortality was 18.5%.The data describe the care of critically ill non-traumatic patients in the resuscitation room. Based on these data, algorithms for the structured care of critically ill non-traumatic patients need to be developed.The online version contains supplementary material available at 10.1186/s12873-023-00812-y. The incidence of emergency medical services (EMS) rescue missions is continuously increasing in Germany nationwide : A complBoth traumatic and non-traumatic emergencies are treated in out-of-hospital setting, with the emergency department (ED) providing the interdisciplinary and integrative interface between out-of-hospital and early inpatient care for patients with life-threatening conditions of any cause , 3. In Gwww.traumaregister.de), which has been established since the year 1997, provides a comprehensive evaluation of the care of severely injured patients.For trauma patients, there is a transsectoral and structured care in Germany with clear guidelines for admission in the ED resuscitation rooms and the corresponding in-hospital follow-up care. The trauma registry of the German Society for Trauma Surgery  are only available for a few specific conditions , but not for the broad mass of unselected critically ill non-traumatic patients. Therefore, epidemiological data for the transsectoral care of critically ill non-traumatic patients are scarce .Unfortunately, there are few studies in Germany that have addressed the epidemiology and care of critically ill non-traumatic patients in the ED  all consecutive critically ill non-traumatic patients admitted to the resuscitation room of the ED of the tertiary-care University Hospital Duesseldorf, Germany from 01 March 2018 to 28 February 2019 were identified and analyzed. The study protocol was approved by the Ethics Committee of the Medical Faculty of the Heinrich Heine University of Duesseldorf, Germany (No. 2020 − 960).In this retrospective single-center cohort study  were recorded. As predefined target ranges of vital signs as surrogate parameters were 100–150 mmHg for systolic blood pressure (SBP), heart rate (HR) between 50 and 100 bpm, and oxygen saturation (SpO2) above 94%.Out-of-hospital EMS management includes prior notification before ED admission using the ABCDE approach. Prior airway management, ventilation, and administration of catecholamines are also included here. The EMS protocol includes the qualifications of the personnel  admitting patients to the ED. The risk score based on the National Advisory Committee of Aeronautics (NACA) score, the preliminary diagnosis, the leading ABCDE problem, and any medical interventions were taken from the EMS protocol. The American Society of Anesthesiologists (ASA) score was determined retrospectively.The out-of-hospital cardiac arrest (OHCA) reporting follows the Utstein definition as closely as possible .During management in the resuscitation room, definitive time points of events and interventions were recorded in the PDMS . The outcomes studied were length of stay (LOS) in the resuscitation room, LOS in the ED, allocation to the intensive care unit and all-cause mortality at day 30.To provide a good overview to the already published studies, we make a comparison between the current OBSERvE-DUS study and three other studies from other sites , 6, 7. Tp < 0.05. Microsoft® Office 365  and DataGraph 5.0  were used for statistical analyses and to prepare figures.Data are presented as numbers and percentages, mean ± standard deviation (SD), median with minimal and maximal values as appropriate. The chi square-test, the student’s t-test, and the Wilcoxon rank-sum test were used to compare groups as appropriate. All tests used were twosided, and statistical significance was set at During the study period, a total of 40,764 patients were treated in the ED, of whom 23,235 patients (57.0%) were admitted for non-traumatic emergencies. Out of all ED patients, 5,206 patients were treated in one of the four available resuscitation rooms, and 1,233 patients met at least one criteria of the resuscitation room admission list from which medical records were manually screened. Of these, 630 critically ill non-traumatic patients  received medical treatment in the ED resuscitation room because of significant ABCDE problems. Nine patients were excluded due to incomplete records due to IT issues caused delayed documentation of each procedure and times could not be accurately tracked, procedures were not documented, or late data entry occurred with inconsistent times. Data from 621 patients  were available for the final analysis.The main findings on patient characteristics, including the leading ABCDE problem, are shown in Table The mean age of the patients was 70 ± 16 years. Women and men were equally distributed . Men were significantly younger than women . The proportion of women aged 80 years and older was significantly higher than that of men . Patient were admitted outside (16:00 and 07:59 h) and within (08:00 and 15:59 h) the core working hours in 52 vs. 48%, respectively. During the week (70%) and at weekends (30%) an average of 87 ± 15 and 94 ± 11 critically ill non-traumatic patients were admitted to the ED, respectively. Most patients were admitted to the resuscitation room by EMS (93.1%), followed by ED triage of walking emergencies (3.5%), interhospital transport (2.7%), and in-hospital medical emergency teams (0.6%).The frequency of different aspects of emergency care provided by EMS and the ED team in the resuscitation room is shown in Table Of the three vital signs blood pressure, heart rate, and peripheral oxygen saturation, 58, 45, and 34% of patients, respectively, showed vital functions outside the predefined target ranges despite out-of-hospital EMS care Fig. . A totalThe main diagnoses verified at hospital discharge leading to ED admission are listed in Table Of 73 out-of-hospital cardiac arrest (OHCA) patients, 52 patients (71.2%) showed return of spontaneous circulation (ROSC) on admission and 21 patients (28.8%) were admitted under ongoing cardiopulmonary resuscitation (CPR). In twelve additional cases, in-hospital cardiac arrest (IHCA) occurred in the resuscitation room. Of note, the proportion of OHCA in the OBSERvE-DUS study with 11.8% was smaller than in the OBSERvE-1 and 2 study with 17.1 and 19.9%, respectively. The ROSC rate at hospital admission with 71.2% was in the OBSERvE-DUS study higher than in the OBSERvE-1 and 2 study with 60.4 and 47.3%, respectively.Most patients  were transferred to an intensive care unit (ICU), including 64 (10.3%) patients that had to be transferred to another hospital. Prior to ICU admission, 23 patients (3.7%) underwent interventions . A proportion of 13.5% (n = 84) of patients were admitted to normal ward and 5.0% (n = 31) were discharged home. During treatment in the resuscitation room, 37 patients (6.0%) died.The mean LOS in resuscitation room during initial care and mean LOS in ED, including waiting time until transfer to ICU or another ward, was 120 ± 101 and 415 ± 479 min, respectively. Hospital and ICU LOS for patients with and without cardiac arrest did not differ significantly from each other , circulatory failure (C), and respiratory insufficiency (B) leads to life-threatening situations in critically ill non-traumatic patients. Although the incidence is very low at 0.5%, trauma sequelae has always be considered and excluded .In line with previous investigations, we found in the OBSERvE-DUS study the need for a high level of resuscitation room care at all times of the day, especially at the weekend and outside of the core working time . In contA scoring or grading system for the severity of non-traumatic condition in the resuscitation room is still missing. The NACA score was used as surrogate parameter as an established out-of-hospital score for illness and injury severity. A proportion of 86.8% of patients fell into the categories between 4 to 6, indicating that they had already had severe systemic illness by the time of sudden cardiac arrest. In the OBSERvE-1 study and OBSERvE-2 study, a NACA score between 4 to 6 was reported in a comparable amount of 92% and 85.0%, respectively . A studyConsistent with previous studies, the majority of critically ill non-traumatic patients were admitted to the ED resuscitation room by EMS in up to 93.1% of cases , 7. In-hIn line with previous investigations, most patients in the OBSERvE-DUS study had neurological or cardiovascular emergencies , 6, 7. IIn accordance with the critically ill patient collective, a variety of emergency medical measures were applied in the ED resuscitation room. Leading among these measures were life-saving measures to eliminate airway and ventilation problems , and to stabilize the cardiovascular condition . These findings were in line with the results from other studies investigating critically ill non-traumatic patients , 6, 7. ADespite the EMS treatment, a high proportion of patients were still in extremis at ED resuscitation room admission using predefined target areas of blood pressure, heart rate and oxygen saturation as surrogate parameters. The special contribution of resuscitation room care was already demonstrated by optimizing blood pressure, heart rate, and oxygen saturation as surrogate parameters in previous studies . In the The outcome of patients in German ED is unclear. The 30-day mortality rate of patients never suffer from OHCA or IHCA during the resuscitation room course was 12.9% lower as in previous studies 24%) [% 4, 6, , 6, 7. TMoreover, the 30-day survival rate of patients with cardiac arrest in the OBSERvE-DUS study was higher with 45.9% than the results from single-center und registry-based studies , 7, 20.The OBSERvE-1 and − 2 studies and the OBSERvE-DUS study used the same definition of OHCA according to the Utstein style, and were conducted in the same kind of EMS system with paramedics and physicians staffed EMS vehicles. However, in the OBSERvE-DUS study, resuscitation room management was performed in a certified cardiac arrest center, which was not developed and implemented in the OBSERvE-1 and − 2 study sites. The EuReCA ONE registry study included many EMS systems across Europe and also EMS systems with paramedics and paramedic/physician staffed ambulances.An important difference was found comparing the results for LOS in the resuscitation room and LOS in ED in the presented OBSERvE-DUS study with other investigations , 6, 7. TTaking into account all the above points, the patient population of critically ill non-traumatic patients represents high-risk patients in the resuscitation room of an ED and the following points should be developed in the future to optimize patient safety and the treatment outcome of these patients:  establisOur investigations suffer from some limitations. At first, this is a retrospective single-center cohort study. With approximately 40,000 patient contacts, our ED is relatively large in a national comparison. However, our results may not be fully transferable to other locations. The data were extracted retrospectively and not collected simultaneously to the resuscitation room care. There is certainly a documentation bias regarding the interventions carried out, with a possible documentation deficit. Against this background, we assume that considerably more interventions were carried out. Our statistical processing of our extracted data, we deliberately chose to present processed parametric tests. The study results with which we want to compare our data were published only with the parametric data given. In order to create a comparability between the datasets, we have also opted for this.The data describe the care of critically ill non-traumatic patients in the resuscitation room. Critically ill non-traumatic patients suffer from a high mortality rate and require comprehensive diagnostic procedures in the resuscitation room. Life-saving interventions are frequently used. An impressive number of patients require structured and well-organized care in the ED resuscitation room. Based on these data, algorithms for the structured care of critically ill non-traumatic patients need to be developed.Below is the link to the electronic supplementary material.Supplementary Material 1


Introduction: Bloodstream infections (BSI) are a major cause of mortality all over the world. Inappropriate empirical antimicrobial treatment (i-EAT) impact on mortality has been largely reported. However, information on related factors for the election of i-EAT in the treatment of BSI in adults is lacking. The aim of the study was the identification of risk-factors associated with the use of i-EAT in BSI.Methods: A retrospective, observational cohort study, from a prospective database was conducted in a 400-bed acute-care teaching hospital including all BSI episodes in adult patients between January and December 2018. The main outcome variable was EAT appropriation. Multivariate analysis using logistic regression was performed.Results: 599 BSI episodes were included, 146 (24%) received i-EAT. Male gender, nosocomial and healthcare-associated acquisition of infection, a high Charlson Comorbidity Index (CCI) score and the isolation of multidrug resistant (MDR) microorganisms were more frequent in the i-EAT group. Adequation to local guidelines’ recommendations on EAT resulted in 91% of appropriate empirical antimicrobial treatment (a-EAT). Patients receiving i-EAT presented higher mortality rates at day 14 and 30 when compared to patients with a-EAT . In the multivariate analysis, a CCI score ≥3 (OR 1.90 (95% CI 1.16–3.12) p = 0.01) and the isolation of a multidrug resistant (MDR) microorganism (OR 3.79 (95% CI 2.28–6.30), p < 0.001) were found as independent risk factors for i-EAT. In contrast, female gender (OR 0.59 (95% CI 0.35–0.98), p = 0.04), a correct identification of clinical syndrome prior to antibiotics administration (OR 0.26 (95% CI 0.16–0.44), p < 0.001) and adherence to local guidelines (OR 0.22 (95% CI 0.13–0.38), p < 0.001) were identified as protective factors against i-EAT.Conclusion: One quarter of BSI episodes received i-EAT. Some of the i-EAT related factors were unmodifiable  but others  could be addressed to optimize the use of antimicrobials. Bloodstream infections are one of the main causes of morbidity and mortality worldwide  and evenAppropriation of empirical antimicrobial treatment (EAT) has become a difficult decision due to the increase of antimicrobial resistance to antibiotics in the past few years, both in the healthcare and the community . This deInadequate EAT (i-EAT) has been largely associated with an increased mortality, compared with an antibiotic regimen that is adequately active against the organisms causing the infection. Many studies have focused on assessing the impact on mortality of an inadequate empirical antimicrobial treatment not only in specific scenarios, like the intensive care units (ICU) .Despite of the relevance of choosing i-EAT, there is much less evidence on the risk factors associated with this practice. Some studies have focused on concrete patient-related factors  while otTherefore, it is understandable that in the past few years, the efforts of the Antimicrobial Stewardship Programs (ASP) had moved towards the design of EAT local guidelines, considering the possibility of participation of resistant microorganisms , not only in the healthcare setting, but also in community-acquired infections. The use of these guidelines has been associated with appropriate EAT (a-EAT) and thus with reduced mortality .Hence, despite some studies have pointed out risk factors for inappropriate empirical antimicrobial treatment for bloodstream infections in specific settings, information on factors related to i-EAT is still scarce. To address this, the aim of the present study was to identify the risk factors associated with the use of inappropriate empirical antimicrobial treatment in bloodstream infections. We hypothesised that there might be some potentially modifiable factors (prescription-related factors) that could be addressed to optimize the EAT choice.A retrospective, observational, cohort study carried out in a 400-bed teaching hospital in Spain .All consecutive episodes of clinically significant bloodstream infection (BSI) in adults (≥16 years old) have been prospectively recorded since 1989, by the Bloodstream Infection Surveillance Program, a multidisciplinary team which makes a prospective clinical follow-up of all patients with documented bacteremia. All BSI cases identified from this database, between January 2018 and December 2018, were included and retrospectively reviewed by three independent Infectious Diseases consultants. A patient could be included in the study more than once if they have different BSIs at different times. Patients were followed-up for 30 days.The need for informed consent was waived because this is an observational quality control study without any effect on patients’ medical management. STROBE recommendations were followed to strengthen the reporting of the study results  von Elm.in-vitro activity (according to susceptibility data) at recommended doses within the first 24 h after the blood cultures had been performed  appropriation. The administration of an antimicrobial with erformed  was defiOther clinical variables collected included age, gender, comorbidities based on Charlson Comorbidity Index (CCI) ; severitThe acquisition lieu of the infection was recorded based on Friedman et al. definitions : a nosocin-vitro EAT for every of these syndromes according with the most frequent aetiology in our setting. A clinical syndrome which had already been included in the local guidelines, with a specific EAT recommendation was considered as an Antimicrobial Stewardship Program syndrome (ASP-syndrome).Local guidelines for EAT have been developed at our institution since 2012–2013 by the Antimicrobial Stewardship Program (ASP). They include 13 different syndromes. Local guidelines’ recommendations suggest active When the origin of the BSI was uncertain despite of careful examination of the clinical and microbiological data, BSI was considered as unknown origin.Pathogens were considered multidrug resistant (MDR) if they met resistance criteria according to Magiorakos et al.  or if thData were collected from the medical charts and electronic medical records.The recommendations of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) were followed for performing, processing, and interpreting the blood cultures . AntibioU-test for comparison of categorical and continuous variables, respectively. A 2-sided p < 0.05 was considered statistically significant.All statistical analyses were performed using the STATA RELEASE 14 software . Categorical variables are presented using counts and percentages and continuous variables as means and standard deviation (SD) or medians and interquartile range (IQR). Univariate analysis was performed using the chi-squared or Fisher exact test, and the Student t-test or Mann–Whitney p < 0.2), a best subset regression procedure was used to identify the most suitable and parsimonious multivariate model, i.e., the one with the lowest Akaike information criterion, which is a well-known parameter of the goodness of fit of the model  curve.A total of 599 BSI episodes were included during the study period, 453 (75.6%) received a-EAT and 146 (24.4%) i-EAT.p = 0.002 at day 14% and 21.9% vs. 9.5%, p < 0.001 at day 30 respectively). There was no association between i-EAT and mortality. Data of univariate and multivariate analysis for mortality at days 14 and 30 are shown in Mortality at days 14 and 30 was higher in the i-EAT episodes .p < 0.001).According to the previous definitions, 24.2% of isolated microorganisms were considered MDR. Within the episodes in which a-EAT was administered, 82/453 (18.1%) were caused by a MDR microorganism (vs. 62/146 (43.1%) which received i-EAT, p = 0.25 at day 14% and 52% vs. 48%, p = 0.32 at day 30 respectively). Regarding the lieu of acquisition, 32.6% of the BSI episodes caused by a MDR microorganism were community-acquired, 28.5% were nosocomial and 38.9% were HC-A (p < 0.001). Differences in the appropriateness of EAT caused by a MDR microorganism according to the healthcare delivery setting were not found (p = 0.70).Mortality rates in BSI episodes caused by a MDR microorganism were 12.5% at day 14% and 17.4% at day 30 with no statistical differences between i-EAT and a-EAT groups .EAT before microbiologic information was prescribed in 549 BSI episodes (91.7%). Considering BSI episodes with no-EAT (49), 23 (47%) were patients without clinical suspicion of infection; in 18 (36.7%) a delayed-initiating antibiotic strategy was chosen due to the clinical stability of the patients and, in the remaining 8 (16.3%), antibiotics were not prescribed because of limitation of therapeutic effort. When the 26 BSI episodes with no-EAT due to clinical decisions  were excluded, the i-EAT rate decreased to 20.9%. Excluding all the BSI episodes with no-EAT, the i-EAT rate was 17.5%.Among all the episodes, 519 (91.2%) were considered ASP-syndromes. Antibiotics according to local guidelines’ recommendations were prescribed in 282 out of 519 ASP-syndromes (54.3%); among these, 25 (8.9%) received i-EAT. Eleven of these patients (44%) had a BSI caused by a MDR organism but with no identifiable risk factors for a MDR microorganisms.Regarding BSI episodes in which the clinical syndrome had been correctly identified but local guidelines’ recommendations were not followed, in 64 out of 127 episodes (50.3%) patients received unnecessary broad-spectrum antibiotics.p < 0.001).The most frequently used antimicrobials for EAT are shown in The unit of admission for the BSI episodes is described in The multivariable logistic regression model used to assess variables associated with i-EAT is shown in In this cohort study of 599 BSI in adults, EAT was inappropriate in 24.2% cases. We identified that male gender, host comorbidities, incorrect identification of clinical syndrome, lack of adherence to local guidelines and the isolation of MDR microorganisms were independent risk factors for i-EAT.The i-EAT rate in our study (24.2%) was similar to previous studies  and evenEvidence on the risk factors for i-EAT in adult patients with BSI is scarce. Previous studies have tried to point them out, but they either limited the analysis to the impact of MDR , were smp = 0.04) as it was in the study conducted by Mettler et al. (K. pneumoniae strains and 77% in KPC-producing strains) and Kadri (49%) and identified that a BSI episode caused by a MDR has nearly a 4-fold greater probability of receiving i-EAT. Prevalence of MDR was also identified as the main unmodifiable risk factor associated with i-EAT in Kadri’s study . Moreover, in a 2018 meta-analysis, isolation of MDR pathogens was independently associated with i-EAT (OR 1.11 95% CI 1.07–1.15)   but also07–1.15)  but, mos07–1.15)  for whic07–1.15) . Hence tp < 0.001). Recently, the impact of misdiagnoses of infection site previous to antibiotic administration has been analysed , analysed . This stanalysed . Investianalysed , despiteanalysed  so the vanalysed  and 3) Panalysed . Thus, ip < 0.001). This finding agrees with a recent study, with a smaller sample size, but with similar design , they were considered as inadequate . Result r design , which rr design . There ar design . Althougr design . The ideIn 2005 McDonald et al. assessed the specific relationship between HC-A status and ineffective initial therapy . Unlike Escherichia coli was the most frequently isolated pathogen, which is consistent with the most frequent sources of infection where E. coli plays a major role.The most frequent sources of BSI were urinary and intraabdominal infections. Urinary and respiratory tract infections received more frequently a-EAT while intraabdominal infections and catheter-related were more frequent sources among i-EAT episodes. Our study has many strengths: 1) It provides a relevant sample size and identifies modifiable related factors for i-EAT in BSI episodes, which leads to opportunities for improvement in the ASP strategies. Other previous studies had smaller sample sizes or have focused on concrete, unmodifiable factors . 2) All In conclusion, one quarter of this cohort of BSI episodes in adults received i-EAT. Some related factors for the choice of i-EAT for bacteremia were unmodifiable, as male gender, high comorbidity or the isolation of a MDR microorganism. However, some important factors were positively associated with a-EAT, as the correct identification of the clinical syndrome before starting EAT and the adherence to local guidelines for EAT, which generate a-EAT in 91% of BSI episodes. The good news is that these modifiable factors could be addressed by the ASP teams to optimize the use of antimicrobials.


Bacillus spp. are beneficial to plants. In this study, we looked into the antagonistic activity of the B. pumilus 3-19 strain and its derivatives following targeted genome editing. Two peptide genes with antibacterial action, bacilysin (bac) and bacteriocin (bact), and the sigF gene, which encodes the sigma factor of sporulation, were specifically inactivated using the CRISPR-Cas9 system in the genome of B. pumilus 3-19. Antibacterial activity against B. cereus and Pantoea brenneri decreased as a result of the inactivation of target genes in the B. pumilus 3-19 genome, with a noticeable effect against bacilysin. The growth dynamics of the culture changed when the bac, bact, and sigF genes were inactivated, and the altered strains had less proteolytic activity. An asporogenic mutant of B. pumilus 3-19 was obtained by inactivating the sigF gene. It has been proven that bacilysin plays a unique part in the development of B. pumilus 3-19’s antagonistic action against soil microorganisms.Due to their capacity to produce antimicrobial peptides that can prevent the growth of diseases, many  Bacillus, Pseudomonas, and Agrobacterium contribute most to biological control as soil- and plant-associated microbes. The main type of rhizobacteria that form spores that can survive in soil for a long period of time under harsh environmental conditions and are able to secrete metabolites that stimulate plant growth and prevent pathogen infestation belong to the Bacillus species [Bacillus species is their diverse secondary metabolism and ability to produce a wide variety of structurally different antagonistic substances. In the B. subtilis genome, from 4 to 5% of the genes are intended for the synthesis of secondary metabolites capable of producing more than 24 structurally diverse antimicrobial compounds [B. amyloliquefaciens FZB42, which produces secondary metabolites such as bacteriocins, antimicrobial peptides and lipopeptides, polyketides, and siderophores, while also possessing growth-promoting properties for plants [Research on the mechanisms of interaction between microorganisms and plants is advancing as interest in the use of microbes with advantageous qualities in agriculture grows. The positive impact of plant rhizosphere bacteria on plant growth and yield has been shown by numerous researchers ,2,3,4,5.ompounds . Anotherr plants .Bacillus sp. can be divided into two subgroups, depending on the synthesis pathway. The first group includes small microbial peptides that are synthesized by large enzymatic complexes [Brevibacillus brevis produces small cyclic peptides thyrocidin and gramicidin S. At the same time, B. licheniformis synthesizes bacitracin A, which consists of 12 amino acids [B. subtilis, B. pumilus, and B. amyloliquefaciens [2+ [Antimicrobial peptides (AMPs) of omplexes . For exano acids . Anotherefaciens . The bioiens [2+ . It has iens [2+ .The second subgroup of antimicrobial peptides includes ribosomal-synthesized peptides named bacteriocins. They contain 12 to 50 amino acid residues and are usually cationic. Bacteria produce bacteriocins against strains closely related to the producer. This group of peptide antibiotics is heterogeneous, amphiphilic, and/or hydrophobic .B. pumilus 3-19 strains showed that it contains the genes responsible for the synthesis of these antimicrobial peptides [B. pumilus 3-19 genome using the CRISPR-Cas genome editing system will be carried out in order to determine the contribution of each of them to the antagonistic activity of the strain.An in silico analysis of the genome of peptides . In thisArabidopsis roots against the phytopathogenic bacterium Pseudomonas syringae by destroying its cell membrane, while Hinarejos et al. described the efficacy of B. subtilis IAB/BS03 surfactin and iturin A against P. syringae [B. subtilis showed cell wall degradation in Xanthomonas campestris pv. campestris and X. axonopodis pv. citri [B. amyloliquefaciens lipopeptides against bacterial plant pathogens.Studies on biological control of bacterial plant pathogens are generally sparse and cover only a few bacterial species. Many authors have pointed to the benefits of surfactin as a surfactant . Howeversyringae ,15. In av. citri . In addiB. subtilis, many physiological processes, such as sporulation, biofilm formation, competence development, proteolytic enzyme synthesis, and production of antimicrobial peptides, are controlled by a quorum-sensing mechanism (by ComQXPA). The critical level of accumulated extracellular peptide pheromones (ComX), competence factors, and sporulation causes a regulatory response of genes that depends on population density. Studies show that the activation of the bacABCDE operon is tightly regulated by four proteins: AbrB and CodY carry out negative regulation of transcription, while ComA and Spo0A are positive (the main regulator of sporulation entry). Thus, the deletion of the spo0A gene led to a sharp decrease in the synthesis of bacilysin. It has recently been shown that the bacilysin-deleted strain is capable of expressing a wide range of proteins [sigF) gene in the B. pumilus 3-19 genome using the CRISPR-Cas genome editing system and further study the antagonistic activity of the resulting mutant.It has recently been found that in proteins . TherefoB. pumilus 3-19, in this work, deletion mutants for the genes of bacilysin (bac), bacteriocin (bact), and sigma factor sporulation (sigF) will be obtained.Thus, to determine the role of AMPs in the antagonistic activity of The strains used in the work are presented in Plasmid pJOE9282.1 was used in the work  .LB agar, LB broth, and Spizizen minimal nutrient medium  were used as nutrient media . Deep cuE. coli DH5α cells was performed using CaCl2 [B. pumilus 3-19 cells were transformed using AEB1 electroporation buffer pH 6.0 [Transformation of recombinant DNA into ng CaCl2 . B. pumir pH 6.0 .The antibiotics streptomycin and kanamycin were used at concentrations of 500 µg/mL and 15 µg/mL, respectively .The dynamics of the growth of wild and mutant strains was carried out for 83 h in 500 mL flasks at a temperature of +37 °C with a swing of 180 rpm.Targeted gene inactivation was carried out at 30 °C for 2–3 days on LA  medium sCRISPR-Cas9  genome editing technology is a modern method for making changes to the genome of bacteria, plants, various cell lines, individual cells, and tissues. Since 2012, the basic CRISPR-Cas9 technology based on Streptococcus pyogenes bacteria has been developed and widely used . The sysB. subtilis genome [E. coli) and pE194ts (temperature-sensitive origin for B. pumilus), as well as the kanamycin resistance gene (kanR). The cas9 gene is under the control of the xylose-inducible promoter (Pxyl), and sgRNA synthesis is regulated by the PvanP promoter [Joseph Altenbuchner presented a single plasmid system that allows efficient editing of the s genome . The shuE. coli DH5α cells and containing the CRISPR/Cas9 system, for further inactivation of target genes [In this work, we used the shuttle vector pJOE9282.1, cloned in et genes . Plasmidbac-/locus_tag = “GZ55_04230”), bacteriocin (bact-/locus_tag = “GZ55_09505”), and sigma factor sporulation (sigF-/locus_tag = “GZ55_10725”).Gene numbers and their sequences were taken from the genome deposited in the database under the numbers: bacilysin , Antarctic Phosphatase , and T4 DNA ligase .http://www.rgenome.net/, accessed on 25 April 2021). To write primers to sgRNA, 20 bp was extraced before or after the selected PAM motif. The BsaI restriction site was additionally added to the sgRNA primers. Primers for R and L fragments of genes were made in Primer-Blast program .The gene sequence for searching of the PAM motif was loaded into the Cas-Designer program . The sequences of primer pairs used are presented in PCR to obtain flanking target gene sequences from The resulting gene fragments were inserted into the pJOE9282.1-sgRNA vector at the SfiI site. Construct integrity was verified by sequencing using T7promoter-For and sgRNA-Rev primers for each construct, respectively. Clone Manager 9 software was used to process the sequencing results.B. pumilus 3-19 for the presence of the resulting constructs containing the CRISPR/Cas9 system  at a wavelength of λ = 590 nm. The amount of biomass was expressed in units of optical density.Proteolytic activity was determined by the breakdown of azocasein  at a concentration of 10 mg/mL dissolved in 0.05 M Tris-HCl buffer, pH 7.3  .The number of spores was counted using the Dorner method (A1) for staining endospores . The numAntimicrobial activity was determined by the method of perpendicular strokes . The test-test analysis, analysis of variance (ANOVA) [p < 0.05 representing a significant difference. All analyses were performed on three biological replicates. The obtained data were processed using Statgraphics Plus 5.0. and GraphPad Prism 8 statistical software and are presented as the mean ± standard deviation (SD). Student’s  (ANOVA) , and TukE. coli DH5α cells and linearized with BsaI.Plasmid pJOE9282.1 (~9059 bp) was isolated from Next, plasmids carrying fragments of the genes of bacilysin, bacteriocin, and sporulation factor sigma-F were constructed. At the first stage, pJOE9282.1 vector and prehybridized spacer fragments (sgRNA) were ligated.B.pumilus 3-19 genomic DNA were inserted into the cloned pJOE9282.1-sgRNA vectors at the SfiI site (bac (L~404 bp and R~402 bp), bact (L~501 bp and R~503 bp), and sigF (L~500 bp and R~716 bp)  .bac), bacteriocin (bact), and sporulation factor (sigF) genes, respectively, were cloned in E. coli DH5α cells. Transformed E. coli DH5α cells were measured for the presence of plasmids pDIb11.21, pVYb11.21, and pGAs11.21 using PCR analysis.The resulting plasmids, pDIb11.21, pVYb11.21, and pGAs11.21, with fragments of the bacilysin  via targeting gRNA and creates a double-strand break. Further, chromosomal DNA undergoes the HDR (homology-directed repair) repair mechanism, or homologous recombination. This mechanism requires the presence of a DNA template . In the bac-L and bac-R (pDIb11.21), fragments of the bacteriocin gene bact-L and bact-R (pVYb11.21), and fragments of the sporulation factor gene sigF-L and sigF-R (pGAs11.21).As a result, we obtained plasmids containing the CRISPR/Cas9 system, target DNA sequence (protospacer), and a DNA template for homologous repair—fragments of the bacilysin gene B. pumilus 3-19 cells using an optimized electroporation method [The resulting plasmids  were transformed into n method . TransfoThe transformation efficiency in the case of the pDIb11.21 vector was ~121 transformants/μg DNA, that of the pVYb11.21 vector was ~155 transformants/μg DNA, and that of the pGAs11.21 vector was ~119.45 transformants/μg DNA.bac, bact, and sigF genes, which was carried out at 30 °C on LA medium with the addition of streptomycin and 0.2% xylose. Colonies appearing within 2 days were placed on LA medium without antibiotic and incubated at 42 °C. Increasing the temperature leads to the loss of the plasmid. This is due to the fact that this plasmid has a temperature-sensitive origin of replication pE194ts. The resulting colonies were selected for subsequent isolation of genomic DNA required for PCR using the following pairs of primers: bac-L-For-bac-R-Rev; bact-L-For-bact-R-Rev; sigF-L-For-sigF-R-Rev. The results are shown in The selected colonies of transformants were further used for inactivation of the sigF gene region in the genome of B. pumilus 3-19 was 1751 bp; after editing the genome, the length of the region was 1216 bp  and ~806 bp (bac) corresponded to the lengths of the gene fragments that are contained in the plasmids pVYb11.21  strains (P. brenneri 3.1 (the zone of inhibition was 1.0 cm).Determination of antimicrobial activity in mutant strains of  sources . The stu strains . The maxbac and bact) resulted in a decrease in antimicrobial activity against B. cereus and P. brenneri . At the same time, it was noted that the antagonistic activity of the Δbac mutant strain (bacilysin gene removed) was lower compared to the Δbact mutant strain (bacteriocin gene removed). Apparently, the contribution of the antimicrobial peptide bacillysin to the manifestation of the antagonistic activity of B. pumilus 3-19 is greater than that of bacteriocin.The spectrum of antimicrobial activity of the tested mutant strains changed. In particular, inactivation of the genes of antimicrobial peptides  led to a sharp decrease in bacilysin synthesis [sigF mutation has minimal effect on the transcriptome under vegetative conditions: 117 genes were expressed differently in the sigF mutant strain compared to the wild-type strain; in particular, the bacteriocin gene was among the genes with reduced expression. The level of its expression in the mutant decreased by 2.67 times compared to the wild strain [sigF was inactivated. The mutant strain ΔsigF was found to be asporogenic. The dynamics of sporulation of the control strain B. pumilus 3-19 is characterized by the presence of spores from the 25th hour onwards; the maximum number of spores was 19%, at the 47th hour  decreased by 42, 37, and 27%, respectively  was close to the growth dynamics of B. pumilus 3-19, and the maximum number of cells was reached at the 33rd hour of culture growth (OD590 = 2.09).The growth dynamics of for 83 h . As a reB. pumilus 3-19 (Δbact) strain, the lag phase was prolonged and ended by the 15th hour of cultivation. The biomass of B. pumilus 3-19 (Δbact) reached its maximum value at the 27th hour (OD590 = 1.52) and was significantly lower (by 29%) compared to the wild strain. Further, the culture growth enters the stationary phase.In the mutant B. pumilus 3-19 mutant in the sigF gene was characterized by an exponential phase lasting 31 h, after which, the stationary phase began. The maximum value of OD590 (1.85) was reached at the 41st hour of cultivation. The cell death phase began at the 65th hour. The growth dynamics of the mutant strain is characterized by a longer stationary phase (The growth dynamics of ry phase .B. pumilus 3-19 showed the highest level of proteolytic enzyme activity at the 65th hour of cultivation (0.498 U/mL). The extracellular enzyme activity in the bac and bact mutant strains was 0.027 U/mL and 0.291 U/mL, respectively, lower than in the wild type. We have shown that the secretion of proteinases in B. pumilus 3-19 decreases upon inactivation of the antimicrobial peptide genes. For the mutant strain ΔsigF, the maximum proteolytic activity occurred by 65 h and was 0.280 U/mL. This indicates a decrease in the proteolytic activity of the mutant strain by 53%.The synthesis of extracellular proteinases by bacilli is related to peptide synthesis and sporulation . In ordesigF, unwanted sporulation is avoided for various experiments such as industrial fermentation, long-term experimental evolution, or null growth studies. There is evidence that disruption of spoIIAC (sigF) increases resistance to spore formation and increases secretion of β-cyclodextrin-glycosyltransferase into the extracellular environment in B. subtilis [B. licheniformis mutant with the removal of sigF [By mutating subtilis . Zhou et of sigF .Thus, in our study, inactivation of target genes resulted in a decrease in proteolytic activity in mutant strains compared to the wild type strain.Since the antimicrobial peptides produced by bacilli are known to be able to limit the growth or activity of pathogenic bacteria, these strains may be useful in the bacterial–plant relationship .B. pumilus 3-19 strain was studied for its antagonistic activity against soil bacteria including phytopathogens. In order to determine the role of a specific antimicrobial peptide synthesized by the B. pumilus 3-19 strain, the genes for antimicrobial peptides (bacilysin (bac) and bacteriocin (bact)) and the gene for the second stage of sporulation spoIIAC (sigF), encoding the sigma factor F, were inactivated in its genome. In this work, the bac-L and bac-R), bacteriocin (bact-L and bact-R), and sporulation factor (sigF-L and sigF-R), respectively. The resulting vectors were used for transformation of B. pumilus 3-19 cells. Targeted inactivation of the bacilysin (bac), bacteriocin (bact), and sporulation factor (sigF) genes in the B. pumilus 3-19 genome was carried out using editing by CRISPR/Cas9 technology.Based on the shuttle vector pJOE9282.1, plasmids pDIb11.21, pVYb11.21, and pGAs11.21 were constructed to carry components of the CRISPR/Cas9 system, spacer fragments (sgRNA), and target regions of target genes, bacilysin , as well as a decrease in antimicrobial activity in relation to B. cereus and P. brenneri . It was noted that, apparently, the contribution of the antimicrobial peptide bacilysin to the manifestation of the antagonistic activity of B. pumilus 3-19 is greater than that of bacteriocin.It was found that the inactivation of antimicrobial peptide genes in the (sigF). The proteolytic activity of the recombinant strain B. pumilus 3-19 (ΔsigF) was 53% lower than that of the native strain; however, it was preserved. This strain exhibited antagonistic activity similar to the mutant strain in the bac gene, indicating that the deletion of the sigF gene led to a decrease in the synthesis of bacilysin.An asporogenic mutant strain was obtained by inactivating the sigma factor gene E. amylovora and P. syringae, antagonistic activity was not manifested in any of the studied strains of B. pumilus 3-19. However, under the influence of the native strain B. pumilus 3-19 and the Δbact mutant, growth retardation was observed in both phytopathogens. This indicates a special role of B. pumilus 3-19 bacilysin in its antagonistic activity against soil bacteria.In relation to the phytopathogens In the future, we plan to elucidate the mechanism of the pleiotropic action of peptides of different natures as a signal molecule, describe the physiological and metabolic processes that these molecules act on, and conduct a comparative characterization of phenotypes and transcriptomic analysis of mutant strains in order to use targeted compositions with a more pronounced antimicrobial effect in practice.